QuantiTect Multiplex PCR Kits

For multiplex, real-time PCR and RT-PCR using sequence-specific probes

Multiplex analysis with no need for optimization

Sensitive detection of as few as 10 copies of each target

Reliable quantification of target and reference genes

Detection of up to 5 targets in the same tube

QuantiTect Multiplex PCR Kits enable reliable quantification of up to 5 gDNA or cDNA targets in a single tube by multiplex, real-time PCR or two-step RT-PCR. The combination of a hot start and a unique PCR buffer system in the ready-to-use master mix ensures highly sensitive and reliable multiplex qPCR on any real-time cycler without the need for optimization. The dNTP mix includes dUTP, allowing optional treatment with UNG. Two kit formats are available: the QuantiTect Multiplex PCR Kit for cyclers that require ROX dye for fluorescence normalization, and the QuantiTect Multiplex PCR NoROX Kit for all other cyclers. For convenience, the master mix can be stored at 2–8°C.

Dilutions of cDNA template (100 ng, 11.11 ng, 1.23 ng, and 0.14 ng) were analyzed by triplex PCR (colored curves) and by singleplex PCR (gray curves) on the iCycler iQ. TaqMan probes labeled with FAM, HEX, or Cyanine 670 dye were used. (Data kindly provided by the University of Minnesota, Minneapolis, MN, USA.)|GAPDH, 18S, HSP, and H28S sequences were amplified either together in 4-plex PCR or in singleplex PCRs on the Rotor-Gene 3000 system using the QuantiTect Multiplex PCR NoROX Kit. The templates were 100, 10, 1, and 0.1 ng of human leukocyte cDNA. Reactions were performed in triplicate. TaqMan probes labeled with FAM, HEX, Texas Red, or Cyanine 670 dye were used. 4-plex PCR and singleplex PCR data are displayed on the amplification plots, with the curves for the singleplex PCRs colored in gray. The plots show reliable multiplex quantification over a wide range of template amounts as well as good reproducibility within each set of triplicates. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; 18S: 18S rRNA; HSP: a heat shock protein; H28S: 28S rRNA.|Three sequences (CSBG, GAPDH, and HSP) were amplified either together in triplex PCR or in singleplex PCRs on the LightCycler 2.0 system using the QuantiTect Multiplex PCR NoROX Kit. Three template mixes with varying amounts of human genomic DNA (for CSBG amplification), linearized plasmid (for GAPDH amplification), and human leukocyte cDNA (for HSP amplification) were prepared. Reactions were performed in duplicate. TaqMan probes labeled with FAM, HEX, or Texas Red dye were used. The amplification plot shows detection of CSBG in the triplex PCRs (Blue: 1400 copies; Green: 140 copies; Red: 14 copies).|[A] Duplex PCR was performed on the ABI PRISM 7700 using either the QuantiTect Multiplex PCR Kit or a kit from Supplier AII. Variable amounts of t(8;14) translocation sequence (50,000, 5000, 500, 50, or 10 copies) were coamplified with a constant amount of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) sequence (107 copies). The t(8;14) translocation sequence was amplified from Ramos cell line genomic DNA using a FAM labeled TaqMan probe. The GAPDH sequence was amplified from a plasmid containing GAPDH cDNA sequence using a HEX labeled TaqMan probe. In contrast to the kit from Supplier AII, the QuantiTect Multiplex PCR Kit reliably quantified high to low amounts of t(8;14) translocation sequence (down to 10 copies, indicated with an arrow) in the presence of a high amount (107 copies) of GAPDH sequence. [B] The procedure described in [A] was repeated, with the exception that the target sequences were amplified separately in singleplex PCRs.|QuantiTect Multiplex PCR Kits provide a simple procedure for quantitative, multiplex, real-time PCR. In contrast to current methods, the kits eliminate the need for optimization of the concentrations of primers, Mg2+, and Taq DNA polymerase, even for difficult assays (e.g., assays in which the copy number of the target gene is much smaller than that for the reference gene).|[A] NH4+ ions prevent nonspecific primers from annealing to the template. [B] Synthetic Factor MP, an innovative PCR additive, increases the local concentration of primers at the template. Together with K+ and other cations, synthetic Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq DNA Polymerase.|

Three sequences were amplified either together in triplex PCR or in singleplex PCRs on the ABI PRISM 7900 using the QuantiTect Multiplex PCR Kit. The templates were 140 pg of genomic DNA from the Ramos cell line carrying a t(8;14) translocation (about 20 copies of the target sequence); 106 copies of a linearized plasmid containing human GAPDH cDNA sequence (glyceraldehyde-3-phosphate dehydrogenase); and 105, 104, or 103 copies of a plasmid containing human NFKB cDNA sequence (nuclear factor of kappa light polypeptide gene enhancer in B-cells). This simulates quantification of low, high, and variable amounts of target genes in a single assay. TaqMan® probes labeled with FAM, HEX, or Bodipy TMR reporter dye plus BHQ quencher were used.

Three sequences (CSBG, GAPDH, and HSP) were amplified either together in triplex PCR or in singleplex PCRs on the LightCycler 2.0 system using the QuantiTect Multiplex PCR NoROX Kit. Three template mixes with varying amounts of human genomic DNA (for CSBG amplification), linearized plasmid (for GAPDH amplification), and human leukocyte cDNA (for HSP amplification) were prepared. Reactions were performed in duplicate. TaqMan® probes labeled with FAM, HEX, or Texas Red dye were used. The table shows the approximate copy numbers of the targets and the mean threshold cycle (CΤ) values for the triplex and singleplex PCRs. The data demonstrate comparable CΤ values between the triplex and singleplex PCRs as well as detection of as little as 14 copies of CSBG sequence in the presence of a high amount (106 copies) of GAPDH sequence. CSBG: Candida solubilized β-glucan; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HSP: a heat shock protein.

Principle

QuantiTect Multiplex PCR Kits enable success in multiplex, two-step RT-PCR on the first attempt (see flowchart "QIAGEN multiplex kits"). The optimized master mix ensures that PCR products in a multiplex reaction are amplified with the same efficiency and sensitivity as PCR products in a corresponding single-amplification reactions. As few as 10 copies of a target gene can be detected with the kit.

Amplifying reference and target genes in the same reaction instead of in separate reactions increases the reliability of gene quantification by minimizing handling errors. The QuantiTect Multiplex PCR Master Mix contains a balanced combination of K+ and NH4+ ions as well as the unique synthetic Factor MP, which together promote stable and efficient annealing of primers and probes to the nucleic acid template, enabling high PCR efficiency (see figure "Unique PCR buffer"). In addition, HotStarTaq DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.

Precise quantification on cyclers that require ROX dye. Does not interfere with reactions on other real-time cyclers

* ROX dye is either present in the master mix or not. See table "Choosing the right QuantiTect Multiplex PCR Kit".

Procedure

QuantiTect Multiplex PCR Kits contain ready-to-use master mixes that eliminate the need for optimization of reaction and cycling conditions. The handbook contains a single protocol that can be used with all available real-time cyclers and also lists recommended dyes. If required, reactions can be pretreated with uracil-N-glycosylase (UNG) (not supplied) to eliminate carryover of PCR products from previous reactions.

Kits are available with or without ROX passive reference dye in the master mix, enabling use on virtually any real-time cycler (see table). Due to the optimized ROX concentrations, detection of even low copy numbers is achieved through automatic data analysis.

Choosing the right QuantiTect Multiplex PCR Kit

ROX dye

Kit

Compatible cyclers

Supplied in master mix

QuantiTect Multiplex PCR Kit

Cyclers from Applied Biosystems

Absent from master mix

QuantiTect Multiplex PCR NoROX Kit

Rotor-Gene cyclers, and cyclers from Bio-Rad, Cepheid, Eppendorf, Roche, Agilent, and other suppliers

QuantiTect Multiplex PCR Kits can be used for gene expression analysis of cDNA or quantification of gDNA on any real-time cycler. This includes instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Agilent. For the Rotor-Gene Q and other Rotor-Gene cyclers, we recommend using the Rotor-Gene Multiplex PCR Kit, which has been specially developed for fast cycling on these instruments.